Parasexuality in fungi is believed by many to be a rare and insignificant process. On the contrary, we have observed that the process occurs regularly and may account for significant genetic exchange on asexual fungi. Asexual pathogens may use parasexuality to generate genetic diversity without disrupting favorable allele combinations. This would have important implications in agriculture and medicine since it may facilitate the transfer of genes for resistance to multiple fungicides or drugs. The importance of reevaluating the role and mechanism of parasexuality in asexual pathogens increases as the number of immunocompromised humans experiencing serious fungal infections increases. In addition to transferring drug resistance, parasexuality may transfer host range genes between pathogens thereby altering host range specificity. If horizontal transfer is possible between unrelated fungi, nonpathogenic fungi normally present in the host could acquire genes conferring pathogenicity. Using Fusarium oxysporum f. sp. cubense (Foc), a haploid imperfect fungus associated with the Ascomycota, as our model system we have isolated 106 auxotrophic and drug resistant mutants. By inducing heterokaryon formation between these mutants, we have observed all the stages of the parasexual cycle in Foc. We have created a new and efficient method for form heterokaryons (double pick) which led to our discovery of mutants which can overcome incompatibility barriers to form heterokaryons with strains outside their own vegetative compatibility groups (VCG) and forma species. We have also developed benomyl resistant double auxotroph strains to induce heterokaryon formation with wild type strains (wild type testers). Some of these strains are also capable of overcoming incompatibility barriers. Such results have encouraged us to reevaluate when transfer of nuclear and cytoplasmic genetic information can occur in the parasexual cycle. In addition to our nuclear encoded auxotrophs, we have identified mitochondrial genome types (mitotypes) by amplification of mitochondrial intergenic regions and analysis of restriction fragment length polymorphisms (RFLP) and single strand conformational polymorphisms (SSCP). We propose to use these advances to assess horizontal transfer of nuclear and cytoplasmic genetic information in heterokaryons, to find mutants showing altered heterokaryon formation, and to assess the wild type testers both as a taxonomic tool and as a means to study in planta formation of heterokaryons.